GlycoAnalytics List of Services

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Below is the list of regular services offered by GlycoAnalytics. We offer a complimentary 1/2 hour consulation with the Core Director to discuss your project and needs. If you don't see a catalog item below that fits your needs, please contact us: glycoanalytics@ucsd.edu.

PREPARATIONS

N-linked glycan is removed from glycoprotein samples using recombinant PNGase-F kit (P0709S) from NEB. Released N-glycans are applied to tandemly connected Sep-Pak C18 and PGC cartridge equilibrated in water. PGC bound glycans are eluted with 30% acetonitrile containing 0.1% TFA in water, lyophilized and used for further analysis.

O-linked glycan is released from glycoproteins using reductive beta-elimination method. Samples are incubated with 0.1 M NaOH/1M NaBH4 at 45°C overnight to release O-linked glycans. The samples are neutralized using ice-cold 30% aqueous acetic acid, followed by removal of sodium salts using cation exchange resin. The samples are coevaporated using methanol/acetic acid to remove excess boric acid, finally the samples are passed over Sep-Pak C18 and the flow through are lyophilized and used for further analysis.

The starting material for this procedure is typically tissues. Tissues are sequentially extracted with chloroform: methanol, twice with each of the following ratios 2:1, 1:1 and 1:2. The pooled extracts are dried and used for further analysis.

The starting material for this procedure is typically tissue or cell pellets. Hard tissues are grinded on liquid nitrogen or soft tissues are homogenized on ice bath on protease treatment buffer. Samples are digested overnight with protease at 37°C and centrifuged at 14000rpm for 20 minutes. The supernatant is passed through a DEAE column and the bound glycosaminoglycan (GAG) is eluted with 2M NaCl. The GAG is then desalted on PD10-size exclusion column and purified GAG is lyophilized and used for further analysis.

Harvested bacterial cells is washed by PBS to remove the adhered media. The cells are suspended in water (7mL water per gm or bacterial cell) and sonicated for 1 minute to make the cell-suspension. The cell suspension is stirred on oil/or water bath maintained at 65°C for 10 minutes using a magnetic stirrer. 7mL of 90% phenol is pre-heated to 65°C and added to the aqueous bacterial suspension. The mixture is stirred for 30 minutes at 65°C and immediately cooled below 10°C on an ice-water bath. The reaction mixture is centrifuged at 4000rpm for 40 minutes at 10°C. The top layer containing Smooth LPS is removed carefully and dialyzed against distilled water for 3-4 days with changing of water each day until the smell of phenol is undetectable. The extracted LPS are treated with DNase/RNase and Proteinase K to remove contaminating nucleic acid and proteins respectively. Finally the LPS are precipitated by ultracentrifugation at 120,000g for 6 hours at 5°C.

Harvested bacterial cells are washed with deionized water (once), 90% ethanol (twice), acetone (twice) and di-ethyl ether (once), and dried in vacuum desiccators. The dried cells are grinded to fine powder and reaction mixture in the ratio of 2:5:8 of 90% phenol: chloroform: petroleum-ether (40-60°C fractions) is added (200 mL of reaction mixture for 50 gm of dried cell or equivalent). This cell-solvent mixture is stirred at high speed maintaining the temperature near 10°C for 30 minutes. The extracted material is centrifuged at 3500 rpm for 10 minutes at 10°C. Supernatant is collected and chloroform and petroleum ether is removed from the mixture by rotary evaporation. Rough LPS is precipitated from the phenol layer by careful addition of water dropwise. Precipitated LPS is centrifuged and repeatedly washed with 5:1 Di-ethyl ether: acetone mixture and dried by Nitrogen flush.

Cells are shaken with PBS or 1% aqueous phenol for 3 hours at room temperature and centrifuged at 7000 rpm for 20 minutes at 5°C. Crude CPS is isolated from supernatant by cold absolute ethanol precipitation. CPS is removed by centrifugation at 14000 rpm for 20 minutes at 5°C. The precipitate is treated with Nuclease and Proteinase K and dialyzed against water. Enzyme treated CPS is further purified by size exclusion chromatography or weak anion exchange chromatography.

Cells are pelleted by centrifugation washed once in ice-cold phosphate buffered saline and then lysed by brief sonication using cold 50% acetonitrile. The lysed cells are centrifuged at 16000 rpm for 10 minutes at 4°C to remove insoluble material and the supernatant is dried using speed vacuum. The dried samples are suspended in 10mM ammonium bicarbonate and sugar nucleotides are purified using EnviCarb graphitized carbon columns (Supelco). The samples are loaded in 10mM ammonium bicarbonate solution and the column is washed with 3mL water, finally eluted with 2mL of 25% acetonitrile, 50mM TEAA buffer. Eluate is freeze-dried and stored at -80°C prior to analysis.

MODIFICATIONS

Dry glycan samples are hydrolysed by 4N TFA at 100°C for 4 hours, followed by removal of the acid by dry nitrogen flush. The acid is removed completely by co-evaporation twice using 1:1 isopropanol: water mixture under dry nitrogen flush. Hydrolyzed samples are reduced overnight by sodium borohydride in 1M ammonium hydroxide solution. Excess borohydride is neutralized by 30% acetic acid and boric acid is removed as their methyl borate. Samples are finally treated with 1:1 acetic anhydride: pyridine mixture at 100°C for 1h. Pyridine and acetic anhydride is removed by nitrogen flush and alditol acetate is extracted with dichloromethane and ready to be injected on GCMS.

Samples should be completely dried before performing TMS derivatization. Samples are methanolyzed using 1M methanolic hydrochloride at 80°C for 16h. Excess acid is removed by dry nitrogen flush, and co-evaporated once with dry methanol. Samples are then re-N-acetylated by 4:1:1 methanol: pyridine: acetic anhydride mixture at 100°C for 1h. Reagents are removed by dry nitrogen flush and finally treated with Tri-Sil (Pierce) reagent at 80°C for 30 minutes. The TMS derivatives of the monosaccharides are extracted with hexane and ready for injection on GC-MS.

The starting material for this procedure should be purified and completely dried glycolipids or glycan. Samples are dissolved in dry DMSO and stirred for several hours until the samples are completely dissolved. Sodium hydroxide slurry in DMSO is added followed by addition of methyl iodide and reacted at room temperature for 45mins. The reaction is stopped adding1mL of ice-cold water. The methylated glycans are extracted in using chloroform, dried and used for further analysis.

The PM samples from Mod-003a are hydrolyzed using 4N TFA at 100°C for 6hours. Followed by removal of the acid, reduction using sodium borodeuteride and acetylation as described in Mod-001. The PMAA derivative is then analyzed using GCMS for inter residue linkage analysis.

Dried samples are methanolysed by 1M methanolic HCl at 80°C for 18 hours. The acid is removed by nitrogen flush, half-saturated sodium chloride is added to the samples and fatty acid methyl ester is extracted using chloroform. The chloroform layer is back extracted with water and dried by nitrogen flush. Fatty acid samples are dissolved in hexane and injected on GC-MS. For determination of hydroxyl fatty acids samples are further treated with Tri-Sil reagent and extracted using hexane.

Samples are treated with 2M acetic acid at 80°C for 3 hours followed by removal of excess acid using speed vacuum. Depending upon the sample type hydrolyzed samples may need further purification by 3K MWCO spin filtrations.

De-O-Acetylation of glycans is achieved by treating the samples with base 50mM of Sodium hydroxide at room temperature for 3 hours or by treatment with anhydrous hydrazine. Sodium hydroxide is neutralized by 30% acetic aicd under cold conditioning and the glycan is purified by PD10-size exclusion chromatography. Anhydrous hydrazine treated material is precipitated by cold acetone; the precipitate is washed once more with cold acetone, and dissolved in water and lyophilized.

Glycosaminoglycans are dissolved in CS digestion buffer and reacted with a cocktail mixture of Chondroitinase ABC at 37°C for 18 hours. Following digestion, the mixture is fractionated using a 10,000 MWCO filter to remove enzyme and undigested glycosaminoglycan chains. The flow through is dried and used for further analysis.

Glycosaminoglycans are dissolved in HS-digestion buffer and reacted with mixture of Heparinase I, II and III at 37°C for 18 hours. Following digestion, the mixture is fractionated using a 10,000 MWCO filter to remove enzyme and undigested glycosaminoglycan chains. The supernatant is dried and used for further analysis.

Dried GAG disaccharides are dissolved in aniline and reacted with freshly prepared sodium cyanoborhydride solution in dimethyl sulfoxide: acetic acid mixture (7:3, v/v). Reactions are carried out at 65°C for 1 hours followed by 37°C for 16 hours. The samples are then dried using speed vacuum at room temperature and used for further analysis by LTQ-MS.

Labeling of glycans with 2-Aminobenzamide (2-AB) requires dried glycans. The samples are reacted with 2-AB dissolved in cyanoborohydride reaction mixture at 65°C for 2.5 hours. Excess reagent was removed from the samples using Glycoclean S-cartridge labelled glycans were dried on a speed vacuum and used for further characterization.

COMPOSITIONS

Measured amount of sample (1-20µg) are treated with 100μl of 2 M TFA at 100°C for 4h followed by removal of excess acid by dry nitrogen flush. To ensure complete removal of acid the samples are co-evaporation twice with 1:1 isopropanol. Finally, the samples are dissolved in water and analyzed by HPAEC-PAD using a CarboPac PA-1, PA-10 or PA-20 column.

Measured amount of sample (10-30µg) are treated with 2M TFA at 100°C for 6 hours followed by removal of excess acid by dry nitrogen flush. To ensure complete removal of acid the samples are co-evaporation twice with 1:1 isopropanol. Finally, the samples are dissolved in water and analyzed by HPAEC-PAD using a CarboPac PA-1.

The sample is dissolved in a final concentration of 2 M HOAc and heated to 80°C for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 MWCO filter, dried and analyzed by HPAEC-PAD using a Dionex CarboPac PA-1 or PA-20 column. Solvent gradient of sodium acetate is used to separate N-acetylneuraminic acid and N-glycolylneuraminic acid.

The starting material for this procedure can be glycoprotein, glycolipid or glycan. Oligosaccharides and glycoconjugates may contain covalently attached non- carbohydrate groups; among those, sulfate and phosphate are quite common. The presence and quantity of those groups is determined by subjecting the sample to pyrolysis, followed by autosampler injection and separation of the anions on an ion-exchange (Dionex IonPac) column. Conductivity detector is used in conjunction with a suppressor to get optimum results. Blanks (that represent background from the methodology used for sample preparation) are required and it is recommended that samples be run in duplicate for optimum quantitative results.

The samples are hydrolyzed using 2 M HOAc at 80°C for 3 hours to release sialic acids. The released sialic acids are purified by spin filtration using 3K MWCO filter and tagged with DMB reagent. The fluorescent tagged sialic acid derivatives are then analyzed by reverse-phase HPLC with on-line fluorescence detector.

Trimethylsilyl derivatives of methyl glycosides of neutral, acidic and basic monosaccharides are analyzed by GC-MS using Restek-5ms capillary column. TMS sugars are detected from their retention time and by electron impact (EI) mass fragmentation spectrum.

Alditol acetate derivative of neutral and amino sugars are separated on Restek-5ms capillary column. AA sugars are detected from their retention time and by electron impact (EI) mass fragmentation spectrum.

Fatty acid methyl esters are dissolved in hexane and analyzed by GC-MS using Restek-5ms capillary column. Identifications are achieved by using a combination of retention time and EI mass fragmentation pattern.

Glycan samples are separated on suitable column and connected online to the mass spectroscope. Acetonitrile: water mixture containing ammonium formate or formic acid can be used as mobile phase for amino-bonded column and methanol: acetonitrile and water can be used for reverse phase columns. Several ion-pairing reagents compatible with mass spectrometry can also be used, optimization required for particular sample.

The mixture of sugar nucleotide is profiled by anion exchange chromatography using a Dionex PA-1 column equilibrated in either water or 10mM sodium hydroxide. Separation of nucleotide sugars are achieved by an increasing gradient of sodium acetate concentration. UV detection is performed at 260 nm and matched with authentic standards at concentration of 0.5-1nmole.

Depending on glycan types samples are dissolved in suitable solvent and mixed with super-DHB matrix solution in 1:1 v/v ratio and spotted on MALDI plate. The samples are dried and analyzed in positive mode or negative mode.

Depending on the nature of the metabolites the samples are analyzed either using Carbo Pak PA-1 or C18 column. Detection is done using UV-detector at 260 nm. Authentic standards are required to identify and quantify the peaks.

It is the policy of GlycoAnalytics Core that each project be given a maximum of 30 minutes of discussion at no charge with the Core Director. Additional time for individualized analysis of the issue, help in selecting the best protocol for the isolation, purification and analysis of glycoconjugates and interpretation of data is available through consultation with the Core Director, depending upon available time.

There may be an additional charge if custom methods are requested or if there is any analysis which is not listed on the website. This charge will not be imposed without prior consultation with the investigator.

GlycoAnalytics Core is always willing to do a method development and discover new strategies for analyzing glycans. This can be done upon request; however, the customer must provide consumables as needed. A fee will be charge for the establishment of a new method and the subsequent transfer of the method to the customer.

GlycoAnalytics Core can do purification of glycans by fraction collection from HPLC for the customer for further analysis; charges will be made according to the time and complexity of the job. Customers are responsible for consumables and shipping charge.

Many of the instruments in GlycoAnalytic Core utilize proprietary software that is not commonly found in most research laboratories. Glyco Analytic Core can therefore provide any data profile in the form of a Microsoft PowerPoint presentation, thus eliminating the need to scan data printouts.

We welcome trainees from other institutions to send trainees to San Diego for more intensive training. You may bring or send your own samples and Core staff will train you to complete your own preparations or analyses. All training must be scheduled in advance with the Core Director.